GM-CSF Variants and Methods of Use

ABSTRACT

GM-CSF variants, polynucleotides encoding them, and methods of making and using the foregoing are useful in treatment of immune-related disorders, such as inflammatory bowel disease (IBD).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/347,342 filed 8 Jun. 2016, U.S. Provisional Application Ser. No.62/374,068, filed 12 Aug. 2016, and U.S. Provisional Application Ser.No. 62/423,857, filed 18 Nov. 2016, the entire contents of theaforementioned applications are incorporated herein by reference intheir entireties.

SEQUENCE LISTING

This application contains a Sequence Listing submitted via EFS-Web, theentire content incorporated herein by reference in its entirety. TheASCII text file, created on 23 May 2017, is named JBI5088USNP_ST25.txtand is 23 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to GM-CSF variants, syntheticpolynucleotides encoding them, and methods of making and using theforegoing

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD) is a disorder of unknown etiologycharacterized typically by diarrhea, cramping, abdominal pains, weightloss and rectal bleeding, tiredness, anemia, fistulae, perforations,obstruction of the bowel and frequent need for surgical intervention.According to the US Center for Disease Control and Prevention, about 1.4million people in USA suffer from IBD, making it one of the mostprevalent gastrointestinal diseases in the United States. The overallhealthcare cost of IBD in USA is estimated to be more than US$1.7billion per year.

A number of disorders fall within the class of IBD, including Crohn'sdisease, ulcerative colitis, indeterminate colitis, microscopic colitisand collagenous colitis. The most common forms of IBD are Crohn'sdisease and ulcerative colitis. Ulcerative colitis affects the largeintestine (colon) and rectum and involves the inner lining (e.g., themucosal and sub-mucosal layer) of the intestinal wall. Crohn's diseasemay affect any section of the gastrointestinal tract (e.g., mouth,esophagus, stomach, small intestine, large intestine, rectum, anus,etc.) and may involve all layers of the intestinal wall. The clinicalsymptoms of IBD include rectal and/or intestinal bleeding, abdominalpain and cramping, diarrhea, and weight loss. In addition, IBD is a riskfactor for colon cancer, and this risk for colon cancer increasessignificantly after eight to ten years of IBD.

IBD has no cure. Current therapies are directed at reducing theinflammatory process and at reducing the detrimental effects of theinflammatory process associated with the disease, and includeadministration of anti-inflammatory drugs (e.g., APRISO® (mesalamine),AZULFIDINE® (sulfasalazine), REMICADE® (infliximab), HUMIRA®(adalimumab), prednisone, budesonide) and of immunosuppressive drugs(e.g., 6-mercaptopurine, azathioprine, cyclosporine). Such therapies maybe associated with adverse side effects, such as nausea, vomiting,anorexia, dyspepsia, malaise, headaches, abdominal pain, fever, rash,pancreatitis, bone marrow suppression, formation of antibodies, infusionreactions, and increased opportunistic infections.

Therefore, a need exists for additional therapies for IBD.

SUMMARY OF THE INVENTION

The invention provides an isolated GM-CSF variant comprising asubstitution S29C and a substitution S69C when compared to the wild typeGM-CSF of SEQ ID NO: 1, optionally further comprising at least onesubstitution at an amino acid residue position corresponding to residueR23, L49 or K107 of SEQ ID NO: 1.

The invention also provides an isolated GM-CSF variant comprising anamino acid sequence of SEQ ID NO: 33.

The invention also provides an isolated GM-CSF variant comprising anamino acid sequence of SEQ ID NOs: 2, 3, 4, 6, 7, 8 or 9.

The invention also provides an isolated GM-CSF variant comprising asubstitution S29C and a substitution S69C when compared to the wild typeGM-CSF of SEQ ID NO: 1, optionally further comprising at least onesubstitution at an amino acid residue position corresponding to residueR23, L49 or K107 of SEQ ID NO: 1, wherein the GM-CSF variant isconjugated to a half-life extending moiety.

The invention also provides an isolated polynucleotide encoding theGM-CSF variant of the invention.

The invention also provides a vector comprising the polynucleotide ofthe invention.

The invention also provides an expression vector comprising thepolynucleotide of the invention.

The invention also provides a host cell comprising the vector of theinvention.

The invention also provides a host cell comprising the expression vectorof the invention.

The invention also provides a method of producing the GM-CSF variant ofthe invention, comprising culturing the host cell of the invention inconditions that the GM-CSF variant is expressed, and purifying theGM-CSF variant.

The invention also provides a kit comprising the GM-CSF variant of theinvention.

The invention also provides a pharmaceutical composition comprising theGM-CSF variant of the invention and a pharmaceutically acceptableexcipient.

The invention also provides a method of treating an inflammatory boweldisease (IBD) in a subject in need thereof, comprising administering tothe subject a therapeutically effective amount of the GM-CSF variant ofthe invention for a time sufficient to treat the IBD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of human GM-CSF (PDB: 2GMF (Rozwarski et al.,1996)) showing the residues considered for engineering to improvestability of GM-CSF.

FIG. 2A shows the amino acid sequence alignments between various GM-CSFvariants from residues 1-60. The number at the beginning of the rowindicates the SEQ ID NO: of the amino acid sequence.

FIG. 2B shows the amino acid sequence alignments between various GM-CSFvariants from residues 61-127. The number at the beginning of the rowindicates the SEQ ID NO: of the amino acid sequence.

FIG. 3 shows the stability of S29C/S69C, L49P, S29C/S69C/K107I andS29C/S69C/R23L/L49P/K107IGM-CSF variants over time (1, 10 and 30 minutesas indicated in the Figure) in fasted state simulated intestinal fluid(FaSSIF) with 3 mg/mL pancreatin. C: control.

FIG. 4A shows that biological activity of the GM-CSF variantsR23L/S29C/L49P/S69C/K107I and S29C/L49P/S69C/K107Iwas retained after 30minute incubation with FaSSIF supplemented with 3 mg/mL pancreatin,whereas the biological activity of the wild-type GM-CSF was completelyabrogated. PP1A7: wild-type GM-CSF, GSFD96:R23L/S29C/L49P/S69C/K107Ivariant; GSFD97: S29C/L49P/S69C/K107I variant.Biological activity was measured in TF-1 cells by assessing percent (%)phosphorylation of Tyr694 of STAT5 and plotted as a function of GM-CSFconcentration used in the assays.

FIG. 4B shows that biological activity of the GM-CSF variantsR23L/S29C/L49P/S69C/K107I and S29C/L49P/S69C/K107I was retained after 1hour of incubation with FaSSIF supplemented with 3 mg/mL pancreatin atcomparable levels to that of the wild-type GM-CSF withoutFaSSIF+pancreatin. PP1A7: wild-type GM-CSF, GSFD96:R23L/S29C/L49P/S69C/K107I variant; GSFD97: S29C/L49P/S69C/K107I variant.Biological activity was measured in TF-1 cells by assessing percent (%)phosphorylation of Tyr694 of STAT5 and plotted as a function of GM-CSFconcentration used in the assays.

FIG. 4C shows that biological activity of the GM-CSF variantR23L/S29C/L49P/S69C/K107I was retained after 4 hours of incubation withFaSSIF supplemented with 3 mg/mL pancreatin at comparable levels to thatof the wild-type GM-CSF without FaSSIF+pancreatin and that the variantS29C/L49P/S69C/K107I demonstrates some activity at this time point.PP1A7: wild-type GM-CSF, GSFD96: R23L/S29C/L49P/S69C/K107I variant;GSFD97: S29C/L49P/S69C/K107I variant. Biological activity was measuredin TF-1 cells by assessing percent (%) phosphorylation of Tyr694 ofSTAT5 and plotted as a function of GM-CSF concentration used in theassays.

FIG. 4D shows that biological activity of the GM-CSF variantR23L/S29C/L49P/S69C/K107I was retained after 6 hours of incubation withFaSSIF supplemented with 3 mg/mL pancreatin. PP1A7: wild-type GM-CSF,GSFD96: R23L/S29C/L49P/S69C/K107I variant; GSFD97: S29C/L49P/S69C/K107Ivariant. Biological activity was measured in TF-1 cells by assessingpercent (%) phosphorylation of Tyr694 of STAT5 and plotted as a functionof GM-CSF concentration used in the assays.

FIG. 5A shows that biological activity of the GM-CSF variantsR23L/S29C/L49P/S69C/K107I and S29C/L49P/S69C/K107I was retained afterincubation for 30 minutes with colon content from naïve cynomolgusmonkeys (CC), whereas the biological activity of the wild-type GM-CSFwas almost completely abolished. PP1A7: wild-type GM-CSF, GSFD96:R23L/S29C/L49P/S69C/K107I variant; GSFD97: S29C/L49P/S69C/K107I variant.Biological activity was measured in TF-1 cells by assessing percent (%)phosphorylation of Tyr694 of STAT5 and plotted as a function of GM-CSFconcentration used in the assays.

FIG. 5B shows that biological activity of the GM-CSF variantsR23L/S29C/L49P/S69C/K107I and S29C/L49P/S69C/K107I was retained afterincubation for 2 hours with colon content from naïve cynomolgus monkeys(CC). PP1A7: wild-type GM-CSF, GSFD96: R23L/S29C/L49P/S69C/K107Ivariant; GSFD97: S29C/L49P/S69C/K107I variant. Biological activity wasmeasured in TF-1 cells by assessing percent (%) phosphorylation ofTyr694 of STAT5 and plotted as a function of GM-CSF concentration usedin the assays.

FIG. 5C shows that biological activity of the GM-CSF variantR23L/S29C/L49P/S69C/K107I was retained after incubation for 6 hours withcolon content from naive cynomolgus monkeys (CC). TheR23L/S29C/L49P/S69C/K107I variant of GM-CSF exhibited a 5-fold loss ofactivity compared to the untreated variant cytokine while the activityof wild-type GM-CSF was completely abolished. PP1A7: wild-type GM-CSF,GSFD96: R23L/S29C/L49P/S69C/K107I variant; GSFD97: S29C/L49P/S69C/K107Ivariant. Biological activity was measured in TF-1 cells by assessingpercent (%) phosphorylation of Tyr694 of STAT5 and plotted as a functionof GM-CSF concentration used in the assays.

FIG. 5D shows that biological activity of GM-CSF and its variantsS29C/L49P/S69C/K107I and R23L/S29C/L49P/S69C/K107I was abolished afterincubation for 24 hours with colon content from naive cynomolgus monkeys(CC). PP1A7: wild-type GM-CSF, GSFD96: R23L/S29C/L49P/S69C/K107Ivariant; GSFD97: S29C/L49P/S69C/K107I variant. Biological activity wasmeasured in TF-1 cells by assessing percent (%) phosphorylation ofTyr694 of STAT5 and plotted as a function of GM-CSF concentration usedin the assays.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

As used herein and in the claims, the singular forms “a,” “and,” and“the” include plural reference unless the context clearly dictatesotherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which an invention belongs. Although any compositions andmethods similar or equivalent to those described herein can be used inthe practice or testing of the invention, exemplary compositions andmethods are described herein.

“Polynucleotide” refers to a molecule comprising a chain of nucleotidescovalently linked by a sugar-phosphate backbone or other equivalentcovalent chemistry. Double and single-stranded DNA and RNA are typicalexamples of polynucleotides.

“Polypeptide” or “protein” refers to a molecule that comprises at leasttwo amino acid residues linked by a peptide bond to form a polypeptide.

“Peptide” refers to a short polypeptide up to 30 amino acids long.

“Vector” refers to a polynucleotide capable of being duplicated within abiological system or that can be moved between such systems. Vectorpolynucleotides typically contain elements, such as origins ofreplication, polyadenylation signal or selection markers that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system, such as a cell, virus, animal, plant, andreconstituted biological systems utilizing biological components capableof duplicating a vector. The vector polynucleotide may be DNA or RNAmolecules or a hybrid of these, single stranded or double stranded.

“Expression vector” refers to a vector that can be utilized in abiological system or in a reconstituted biological system to direct thetranslation of a polypeptide encoded by a polynucleotide sequencepresent in the expression vector.

“Complementary sequence” refers to a second isolated polynucleotidesequence that is antiparallel to a first isolated polynucleotidesequence and that comprises nucleotides complementary to the nucleotidesin the first polynucleotide sequence.

“About” means within an acceptable error range for the value asdetermined by one of ordinary skill in the art, which will depend inpart on how the value is measured or determined, i.e., the limitationsof the measurement system. Unless explicitly stated otherwise within theExamples or elsewhere in the Specification in the context of aparticular assay, result or embodiment, “about” means within onestandard deviation per the practice in the art, or a range of up to 5%,whichever is larger.

“Sample” refers to a collection of similar fluids, cells, or tissuesisolated from a subject, as well as fluids, cells, or tissues presentwithin a subject. Exemplary samples are biological fluids such as blood,serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid,tear drops, feces, sputum, mucosal secretions of the secretory tissuesand organs, vaginal secretions, ascites fluids, fluids of the pleural,pericardial, peritoneal, abdominal and other body cavities, fluidscollected by bronchial lavage, synovial fluid, liquid solutionscontacted with a subject or biological source, for example, cell andorgan culture medium including cell or organ conditioned medium, lavagefluids and the like, tissue biopsies, fine needle aspirations orsurgically resected tissue.

“In combination with” means that two or more therapeutics areadministered to a subject together in a mixture, concurrently as singleagents or sequentially as single agents in any order.

“Subject” includes any human or nonhuman animal “Nonhuman animal”includes all vertebrates, e.g., mammals and non-mammals, such asnonhuman primates, sheep, dogs, cats, horses, cows, chickens,amphibians, reptiles, etc. Except when noted, the terms “patient” and“subject” are used interchangeably.

“Variant” refers to a polypeptide or a polynucleotide that differs froma reference polypeptide or a reference polynucleotide by one or moremodifications, for example one or more substitutions, insertions ordeletions. For example, the variant differs from a wild-type matureGM-CSF polypeptide of SEQ ID NO: 1 or the polynucleotide encoding thewild-type mature GM-CSF having the sequence of SEQ ID NO: 18 by one ormore modifications for example, substitutions, insertions or deletionsof nucleotides or amino acids.

“Isolated” refers to a homogenous population of molecules (such assynthetic polynucleotides or synthetic polypeptides) which have beensubstantially separated and/or purified away from other components ofthe system the molecules are produced in, such as a recombinant cell, aswell as a protein that has been subjected to at least one purificationor isolation step. “Isolated GM-CSF variant” refers to a GM-CSF variantthat is substantially free of other cellular material and/or chemicalsand encompasses variants that are isolated to a higher purity, such asto 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

“Inflammatory bowel disease (IBD)” refers to a disorder or diseasecharacterized by inflammatory activity in the GI tract. IBD includes,but is not limited to, Crohn's disease, ulcerative colitis, Johne'sdisease, Behçet's syndrome, collagenous colitis, diversion colitis,indeterminate colitis, microscopic colitis, infective colitis, ischaemiccolitis, lymphocytic colitis, idiopathic inflammation of the smalland/or proximal intestine, IBD-related diarrhea and closely relateddiseases and disorders of the gastrointestinal tract.

Throughout the specification, residues that are substituted in theGM-CSF variants are numbered corresponding to their position in thewild-type GM-CSF of SEQ ID NO: 1. For example, “S29C” in thespecification refers to the substitution of serine at residue positionthat corresponds to the position 29 in the wild-type GM-CSF of SEQ IDNO: 1 with cysteine.

Abbreviations of natural amino acids are as used herein are shown inTable 1.

TABLE 1 Amino acid Three letter code One letter code Alanine Ala AArginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C GlutamateGlu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V

Compositions of Matter

The present invention provides granulocyte-macrophage colony stimulatingfactor (GM-CSF) variants having enhanced stability and/or biologicalactivity when compared to the wild-type GM-CSF. The present inventionalso provides GM-CSF variants having improved stability in the milieumimicking that of the gastrointestinal track. Therefore, the GM-CSFvariants of the present invention may be suitable for oraladministration.

The GM-CSF variants of the invention may be used in therapeutictreatment of subjects having inflammatory bowel disease (IBD), or otherdiseases or conditions in which potentiation of GM-CSF activity isdesired. The polynucleotides, vectors and cell lines of the inventionare useful in generating the GM-CSF variants of the invention.

GM-CSF is a key regulator of intestinal innate immunity (Däbritz, 2014)and plays a role in the maintenance of intestinal barrier integrity.Recent evidence demonstrates that enteric GM-CSF is also involved inmaintaining tolerance in the mucosa (Mortha et al., 2014). In someCrohn's disease (CD) patients, the etiology has been hypothesized to bea dysregulation of the innate immune system, specifically insufficiencyof neutrophil function and that of other GM-CSF-responsive immune cells(Korzenik & Dieckgraefe, 2000).

Systemic GM-CSF administration has been tested in the clinic for theability to induce remission in patients with CD (Dieckgraefe & Korzenik,2002; Kelsen et al., 2010; Korzenik, 2005; Vaughan & Drumm, 1999).Despite some promising early clinical results, some adverse events werefound in the GM-CSF treatment group in a Phase II CD trial (Valentine etal., 2009). Further, documented increased risk of thrombosis andpulmonary disorders in IBD patients (Bernstein, Blanchard, Houston, &Wajda, 2001; Bernstein, Wajda, & Blanchard, 2008) may also beexacerbated by systemic GM-CSF therapy.

Orally administered GM-CSF for local GI delivery could prove moredesirable from a patient compliance as well as from a safety perspectiveby minimizing systemic exposure. However, oral delivery of protein tothe lower gastrointestinal tract presents challenges owing to the harshpH, proteolytic, and microbial environments to which the biologic drugwould be exposed (Amidon, Brown, & Dave, 2015). Indeed, four-helixbundle growth factors similar to that of GM-CSF have been demonstratedto be rapidly degraded by digestive proteases in vitro (Jensen-Pippo,Whitcomb, DePrince, Ralph, & Habberfield, 1996).

The invention also provides an isolated GM-CSF variant comprising asubstitution S29C and a substitution S69C when compared to the wild typeGM-CSF of SEQ ID NO: 1, optionally further comprising at least onesubstitution at an amino acid residue position corresponding to aresidue R23, L49 or K107 of SEQ ID NO: 1.

The invention also provides an isolated GM-CSF variant comprising anamino acid sequence of SEQ ID NO: 33. SEQ ID NO: 33 is a consensussequence of GM-CSF variant having S29C and S69C substitutions andoptionally substitutions at residue positions R23, L49 or K107.

SEQ ID NO: 33 APARSPSPSTQPWEHVNAIQEAX₁RLLNLCRDTAAEMNETVEVISEMFDX₂QEPTCLQTRLELYKQGLRGCLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFX₃ENLKDFLLVIPFDCWEPVQE;wherein

X₁ is R or L;

X₂ is L or P; and

X₃ is K or I.

The GM-CSF variant comprising the S29C substitution and the S69Csubstitution is more stable and more potent when compared to thewild-type GM-CSF. The substitutions create a novel disulfide bond thatlinks GM-CSF loop AB and loop BC.

Exemplary GM-CSF variants with the S29C substitution and the S69Csubstitution are variants having the amino acid sequence of SEQ ID NOs:2, 6, 7, 8 and 9.

The invention also provides an isolated GM-CSF variant comprising asubstitution S29C and a substitution S69C when compared to the wild typeGM-CSF of SEQ ID NO: 1, optionally further comprising at least onesubstitution at an amino acid residue position corresponding to residueR23, L49 or K107 of SEQ ID NO: 1, wherein the variant exhibits at leastabout 5° C. higher melting temperature (T_(m)) when compared to that ofthe wild-type GM-CSF, wherein the T_(m) is measured using differentialscanning calorimetry using a protocol described in Example 1.

GM-CSF variants having higher T_(m) (e.g. increased thermal stability)are expected to have improved resistant to proteolysis, as it is wellestablished that increased thermal stability generally translates toimproved resistance to proteolysis (Akasako, Haruki, Oobatake, & Kanaya,1995; Daniel, Cowan, Morgan, & Curran, 1982; McLendon & Radany, 1978;Parsell & Sauer, 1989).

The invention also provides an isolated GM-CSF variant comprising asubstitution S29C and a substitution S69C when compared to the wild typeGM-CSF of SEQ ID NO: 1, optionally further comprising at least onesubstitution at an amino acid residue position corresponding to residueR23, L49 or K107 of SEQ ID NO: 1, wherein the variant stimulatesproliferation of TF-1 ATCC® CRL 2003™ cells with an EC₅₀ value that isat least about 1.5-fold less when compared to the EC₅₀ value ofstimulation of proliferation of the TF-1 ATCC® CRL 2003™ cells with thewild-type GM-CSF using a protocol described in Example 1.

GM-CSF variants having a lower EC₅₀ value for their effect in inducingTF-1 cell proliferation when compared to the wild-type GM-CSF in aremore potent activators of GM-CSF signaling pathways.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23A substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23D substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23E substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23F substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23G substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23H substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R231 substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23K substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23L substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23M substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23N substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23P substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23Q substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23S substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23T substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23V substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23W substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue R23 of SEQ ID NO: 1 is a R23Y substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49A substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49D substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49E substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49F substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49G substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49H substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L491 substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49K substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49M substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49N substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49P substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49Q substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49R substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49S substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49T substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49V substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49W substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue L49 of SEQ ID NO: 1 is a L49Y substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107A substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107D substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107E substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107F substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107G substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107H substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107I substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107L substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107M substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107N substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107P substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107Q substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107R substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107S substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107T substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107V substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107W substitution.

In some embodiments, the substitution at the amino acid residue positioncorresponding to residue K107 of SEQ ID NO: 1 is a K107Y substitution.

In some embodiments, the GM-CSF variant comprises a R23L substitution, aL49P substitution and a K107I substitution. These substitutions improvethermal stability and potency of the GM-CSF variant. In addition, theL49P substitution removes a potential MHC class II epitope and thereforethe GM-CSF variants with the L49P substitution may be less immunogenic.

In some embodiments, the GM-CSF variant comprises the S29C substitution,the S69C substitution and the R23L substitution. These substitutionsimprove thermal stability and potency of the GM-CSF variant.

In some embodiments, the GM-CSF variant comprises the S29C substitution,the S69C substitution and the L49P substitution. These substitutionsimprove thermal stability and potency of the GM-CSF variant. Inaddition, the L49P substitution removes a potential MHC class II epitopeand therefore the GM-CSF variants with the L49P substitution may be lessimmunogenic.

In some embodiments, the GM-CSF variant comprises the S29C substitution,the S69C substitution and the K107I substitution. These substitutionsimprove thermal stability and potency of the GM-CSF variant.

In some embodiments, the GM-CSF variant comprises the S29C substitution,the S69C substitution, the R23L substitution and the L49P substitution.These substitutions improve thermal stability of the GM-CSF variant. Inaddition, the L49P substitution removes a potential MHC class II epitopeand therefore the GM-CSF variants with the L49P substitution may be lessimmunogenic.

In some embodiments, the GM-CSF variant comprises the S29C substitution,the S69C substitution, the R23L substitution and the K107I substitution.These substitutions improve thermal stability of the GM-CSF variant.

In some embodiments, the GM-CSF variant comprises the S29C substitution,the S69C substitution, the L49P substitution and a K107I substitution.These substitutions improve thermal stability and potency of the GM-CSFvariant. In addition, the L49P substitution removes a potential MHCclass II epitope and therefore the GM-CSF variants with the L49Psubstitution may be less immunogenic.

In some embodiments, the GM-CSF variant comprises the S29C substitution,the S69C substitution, the R23L substitution, the L49P substitution andthe K107I substitution. These substitutions improve thermal stabilityand potency of the GM-CSF variant. In addition, the L49P substitutionremoves a potential MHC class II epitope and therefore the GM-CSFvariants with the L49P substitution may be less immunogenic.

In some embodiments, the GM-CSF variant comprises a substitution at anamino acid residue position corresponding to residue R23 of SEQ ID NO:1.

In some embodiments, the GM-CSF variant comprises a substitution at anamino acid residue position corresponding to residue L49 of SEQ ID NO:1.

In some embodiments, the GM-CSF variant comprises a substitution at anamino acid residue position corresponding to residue K107 of SEQ ID NO:1.

In some embodiments, the GM-CSF variant comprises a R23L substitution.The substitution improves thermal stability and potency of the GM-CSFvariant.

In some embodiments, the GM-CSF variant comprises a L49P substitution.The substitution improves thermal stability of the GM-CSF variant andremoves a potential MHC class II epitope and therefore the GM-CSFvariants with the L49P substitution may be less immunogenic.

In some embodiments, the GM-CSF variant comprises a K107I substitution.The substitution improves thermal stability of the GM-CSF variant.

In some embodiments, the GM-CSF variant comprises a R23L substitutionand a L49P substitution. These substitutions improve thermal stabilityand potency of the GM-CSF variant. In addition, the L49P substitutionremoves a potential MHC class II epitope and therefore the GM-CSFvariants with the L49P substitution may be less immunogenic.

In some embodiments, the GM-CSF variant comprises a R23L substitutionand a K107I substitution. These substitutions improve thermal stabilityand potency of the GM-CSF variant.

In some embodiments, the GM-CSF variant comprises a L49P substitutionand a K107I substitution. These substitutions improve thermal stabilityand potency of the GM-CSF variant. In addition, the L49P substitutionremoves a potential MHC class II epitope and therefore the GM-CSFvariants with the L49P substitution may be less immunogenic.

In some embodiments, the GM-CSF variant comprises the amino acidsequence of SEQ ID NO: 2.

In some embodiments, the GM-CSF variant is encoded by a polynucleotidecomprising the polynucleotide sequence of SEQ ID NO: 10.

In some embodiments, the GM-CSF variant comprises the amino acidsequence of SEQ ID NO: 3.

In some embodiments, the GM-CSF variant is encoded by a polynucleotidecomprising the polynucleotide sequence of SEQ ID NO: 11.

In some embodiments, the GM-CSF variant comprises the amino acidsequence of SEQ ID NO: 4.

In some embodiments, the GM-CSF variant is encoded by a polynucleotidecomprising the polynucleotide sequence of SEQ ID NO: 12.

In some embodiments, the GM-CSF variant comprises the amino acidsequence of SEQ ID NO: 5.

In some embodiments, the GM-CSF variant is encoded by a polynucleotidecomprising the polynucleotide sequence of SEQ ID NO: 13.

In some embodiments, the GM-CSF variant comprises the amino acidsequence of SEQ ID NO: 6.

In some embodiments, the GM-CSF variant is encoded by a polynucleotidecomprising the polynucleotide sequence of SEQ ID NO: 14.

In some embodiments, the GM-CSF variant comprises the amino acidsequence of SEQ ID NO: 7.

In some embodiments, the GM-CSF variant is encoded by a polynucleotidecomprising the polynucleotide sequence of SEQ ID NO: 15.

In some embodiments, the GM-CSF variant comprises the amino acidsequence of SEQ ID NO: 8.

In some embodiments, the GM-CSF variant is encoded by a polynucleotidecomprising the polynucleotide sequence of SEQ ID NO: 16.

In some embodiments, the GM-CSF variant comprises the amino acidsequence of SEQ ID NO: 9.

In some embodiments, the GM-CSF variant is encoded by a polynucleotidecomprising the polynucleotide sequence of SEQ ID NO: 17.

The GM-CSF variants of the invention may be obtained frompolynucleotides encoding the GM-CSF variants by the use of cell-freeexpression systems such as reticulocyte lysate based expression systems,or by standard recombinant expression systems. For example, thepolynucleotides encoding the GM-CSF variants may be synthesized usingchemical gene synthesis according to methods described in U.S. Pat. No.6,521,427 and U.S. Pat. No. 6,670,127, utilizing degenerateoligonucleotides to generate the desired variants, or by standard PCRcloning and mutagenesis. The polynucleotides encoding the GM-CSFvariants may be cloned into expression vectors and expressed usingstandard procedures. The expressed GM-CSF may be purified using forexample CaptoQ anion exchange, Capto Phenyl HIC resin and DEAE anionexchange. The generated GM-CSF variants may be tested for their improvedthermal stability and potency using assays described for example inExample 1.

Homologous GM-CSF Molecules

Additional substitutions may be made to the GM-CSF variants of theinvention as long as the resulting variants comprise a substitution S29Cand a substitution S69C when compared to the wild type GM-CSF of SEQ IDNO: 1 and retain or have enhanced thermal stability and/or potency whencompared to the parental GM-CSF variant. Thermal stability and potencymay be assessed using the protocols described in Example 1.

Additional substitutions that can be made are those that are earlierdescribed:

-   R24L described in U.S. Pat. No. 5,391,485;-   R23L/N27D/T39E/E123K described in U.S. Pat. No. 5,405,952;-   Q20A and/or E21A described in Int. Patent Publ. No. WO1989/010403;-   substitutions shown in Table 2 and Table 3 and as described in U.S.    Pat. No. 7,208,147.

Conservative modifications may also be made to the GM-CSF variants ofthe invention as long as the resulting variants comprise a substitutionS29C and a substitution S69C when compared to the wild type GM-CSF ofSEQ ID NO: 1 and retain or have enhanced thermal stability and/orpotency when compared to the parental GM-CSF variant.

“Conservative modifications” refer to amino acid modifications that donot significantly affect or alter the characteristics of the moleculecontaining the amino acid sequences. Conservative modifications includeamino acid substitutions, additions and deletions. Conservativesubstitutions are those in which the amino acid is replaced with anamino acid residue having a similar side chain The families of aminoacid residues having similar side chains are well defined and includeamino acids with acidic side chains (e.g., aspartic acid, glutamicacid), basic side chains (e.g., lysine, arginine, histidine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), uncharged polar side chains (e.g., glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine,tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan,histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine,valine, leucine, isoleucine, serine, threonine), amide (e.g.,asparagine, glutamine), beta-branched side chains (e.g., threonine,valine, isoleucine) and sulfur-containing side chains (cysteine,methionine). Furthermore, any native residue in the polypeptide may alsobe substituted with alanine, as has been previously described foralanine scanning mutagenesis (MacLennan et al., (1988) Acta PhysiolScand Suppl 643:55-67; Sasaki et al., (1988) Adv Biophys 35:1-24).

The substitutions may be made individually or combinatorially usingknown methods. For example, amino acid substitutions to the GM-CSFvariants of the invention may be made by known methods for example byPCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, libraries ofvariants may be generated for example using random (NNK) or non-randomcodons, for example DVK codons, which encode 11 amino acids (Ala, Cys,Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting GM-CSFvariants may be tested for their thermal stability and potency usingprotocols described in Example 1.

TABLE 2 Wild- Residue type number residue Possible substitution 16 V A CD E G H K N P Q R S T 19 I A C D E G H K N P Q R S T 25 L A C D E G H KN P Q R S T 26 L A C D E G H K N P Q R S T 28 L A C D E F G H K N P Q RS T 36 M A C D E G H K N P Q R S T 40 V A C D E G H K N P Q R S T 43 I AC D E G H K N P Q R S T 46 M A C D E G H K N P Q R S T 47 F A C D E G HK N P Q R S T 49 L A C D E G H K N P Q R S T 55 L A C D E G H K N P Q RS T 59 L A C D E G H K N P Q R S T 61 L A C D E G H K N P Q R S T 62 Y AC D E G H K N P Q R S T 66 L A C D E G H K N P Q R S T 70 L A C D E G HK N P Q R S T 73 L A C D E G H K N P Q R S T 77 L A C D E G H K N P Q RS T 79 M A C D E G H K N P Q R S T 80 M A C D E G H K N P Q R S T 84 Y CD E G H N P R S T 101 I A C D E G H K N P Q R S T 106 F A C D E G H K NP Q R S T 110 L A C D E G H K N P Q R S T 113 F A C D E G H K N P Q R ST 114 L A C D E G H K N P Q R S T 115 L A C D E G H K N P Q R S T 117 IA C D E G H K N P Q R S T

TABLE 3 Wild- Residue type number residue Possible substitution 15 H A CF G I L M P V W Y 18 A F H K L N P Q R S T W Y 20 Q T 21 E F I P V W Y22 A D E F H I K N P Q R S T V W 24 R A C F G I L M P V W Y 26 L F I M VW Y 31 D H 34 A H K N P Q R S T V W Y 35 E A C G P 36 M W Y 37 N A C G P38 E A C G P 42 V A C D E G H K M N P Q R S T W 45 E A C F G I L M P V WY 47 F W 49 L W Y 50 Q P 60 E A C G P 61 L F I M 63 K A C G I M P Y 64 QA C G P 66 L F I M V 67 R A C G P 69 S T 70 L M W 71 T A C G P 72 K T 74K T 75 G H P 77 L F I W Y 78 T A C G P W Y 82 S A C F G M P V W Y 85 K HP 87 H A C F G I M P W Y 88 C D E H K N P Q R S T W 109 N T 121 C P Y122 W T

Half-Life Extending Moieties

The invention also provides a GM-CSF variant conjugated to a half-lifeextending moiety.

In some embodiments, the half-life extending moiety is a human serumalbumin (HAS), a variant of the human serum albumin, such as a C34Svariant, a transthyretin (TTR), a thyroxine-binding globulin (TGB), analbumin-binding domain, or an Fc or fragments thereof. The half-lifeextending moiety may be conjugated to the N-terminus or to theC-terminus of the GM-CSF variant.

In some embodiments, the half-life extending moiety is conjugated to theN-terminus of GM-CSF.

In some embodiments, the Fc is an IgG1, an IgG2, an IgG3 or an IgG4isotype.

In some embodiments, the half-life extending moiety is a C34S variant ofHSA conjugated to the N-terminus of GM-CSF.

In some embodiments, the half-life extending moiety is a C34S variant ofHSA conjugated to the N-terminus of GM-CSF via a linker of SEQ ID NO:23.

In some embodiments, the half-life extending moiety is a C34S variant ofHSA conjugated to the N-terminus of GM-CSF via a linker of SEQ ID NO:27.

In some embodiments, the half-life extending moiety is a Fc conjugatedto the N-terminus of GM-CSF.

In some embodiments, the half-life extending moiety is a Fc conjugatedto the N-terminus of GM-CSF via a linker of SEQ ID NO: 23.

In some embodiments, the half-life extending moiety is a Fc conjugatedto the N-terminus of GM-CSF via a linker of SEQ ID NO: 27.

In some embodiments, the linker comprises the amino acid sequence of SEQID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

In some embodiments, the Fc comprises at least one substitution. Fcsubstitutions may be made to the Fc to modulate the effector functionsand pharmacokinetic properties of the GM-CSF variant conjugated to theFc.

Fc positions that may be substituted to modulate the half-life of an Fccontaining molecule are those described for example in Dall'Acqua etal., (2006) J Biol Chem 281:23514-240, Zalevsky et al., (2010) NatBiotechnol 28:157-159, Hinton et al., (2004) J Biol Chem279(8):6213-6216, Hinton et al., (2006) J Immunol 176:346-356, Shieldset al. (2001) J Biol Chem 276:6591-6607, Petkova et al., (2006). IntImmunol 18:1759-1769, Datta-Mannan et al., (2007) Drug Metab Dispos,35:86-94, 2007, Vaccaro et al., (2005) Nat Biotechnol 23:1283-1288,Yeung et al., (2010) Cancer Res, 70:3269-3277 and Kim et al., (1999) EurJ Immunol 29: 2819, and include positions 250, 252, 253, 254, 256, 257,307, 376, 380, 428, 434 and 435. Exemplary substitutions that may bemade singularly or in combination are substitutions T250Q, M252Y, I253A,S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A,N434H, N434F, H435A and H435R. Exemplary singular or combinationsubstitutions that may be made to increase the half-life of the Fccontaining molecule are substitutions M428L/N434S, M252Y/S254T/T256E,T250Q/M428L, N434A and T307A/E380A/N434A. Exemplary singular orcombination substitutions that may be made to reduce the half-life ofthe Fc containing molecule are substitutions H435A, P2571/N434H,D376V/N434H, M252Y/S254T/T256E/H433K/N434F, T308P/N434A and H435R.

In some embodiments, the Fc comprises at least one substitution thatreduces binding of the Fc containing molecule to an activating Fcγreceptor (FcγR) and/or reduces Fc-mediated effector functions.

Fc positions that may be substituted to reduce binding of the Fccontaining molecule to the activating FcγR and subsequently to reduceeffector function are those described for example in Shields et al.,(2001) J Biol Chem 276:6591-6604, Intl. Patent Publ. No. WO2011/066501,U.S. Pat. Nos. 6,737,056 and 5,624,821, Xu et al., (2000) Cell Immunol,200:16-26, Alegre et al., (1994) Transplantation 57:1537-1543, Bolt etal., (1993) Eur J Immunol 23:403-411, Cole et al., (1999)Transplantation, 68:563-571, Rother et al., (2007) Nat Biotechnol25:1256-1264, Ghevaert et al., (2008) J Clin Invest 118:2929-2938, An etal., (2009) mAbs, 1:572-579) and include positions 214, 233, 234, 235,236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330,331 and 365. Exemplary substitutions that may be made singularly or incombination are substitutions K214T, E233P, L234V, L234A, deletion ofG236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A,H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F,A330S and P331S in IgG1, IgG2, IgG3 or IgG4. Exemplary combinationsubstitutions that result in Fc containing molecules with reducedeffector functions are substitutions L234A/L235A on IgG1,V234A,/G237A/P238S/H268A/V309L/A330S/P331S on IgG2, F234A/L235A on IgG4,S228P/F234A/L235A on IgG4, N297A on all Ig isotypes, V234A/G237A onIgG2, K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D365E/L358M onIgG1, H268Q/V309L/A330S/P331S on IgG2, S267E/L328F on IgG1,L234F/L235E/D265A on IgG1, L234A/L235A/G237A/P238S/H268A/A330S/P331S onIgG1, S228P/F234A/L235A/G237A/P238S on IgG4, andS228P/F234A/L235A/G236-deleted/G237A/P238S on IgG4. Hybrid IgG2/4 Fcdomains may also be used, such as Fc with residues 117-260 from IgG2 andresidues 261-447 from IgG4.

In some embodiments, the half-life extending moiety is conjugated to theGM-CSF variant via a polypeptide linker. Suitable linkers are forexample linkers shown in Table 4.

TABLE 4 SEQ ID Linker name Linker AA Sequence NO: 1FU1ASLDTTAENQAKNEHLQKENERLLRDWNDVQG 20 RFEKGS 1DC1(13AA)₂ASEKNKRSTPYIERAEKNKRSTPYIERAGS 21 1DC1(13AA)₃ASEKNKRSTPYIERAEKNKRSTPYIERAEKNKRST 22 PYIERAGS AS(AP)₁₀GSASAPAPAPAPAPAPAPAPAPAPGS 23 AS(AP)₂₀GSASAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP 24 APAPAPAPGS (EAAAK)₄ASAEAAAKEAAAKEAAAKEAAAKAGS 25 (EAAAK)₈ ASAEAAAKEAAAKEAAAKEAAAKEAAAKEAAA26 KEAAAKEAAAKAGS GS(G₄S)₄ GSGGGGSGGGGSGGGGSGGGGS 27 GS(G₄S)₈GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 28 GGGGSGGGGS GS12X(G4S)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 29 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGS16X(G4S) GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 30GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGS

In some embodiments, the half-life extending moiety is an ethyleneglycol, a polyethylene glycol (PEG) molecule, such as PEG5000 orPEG20000, a dextran, a polylysine, fatty acids and fatty acid esters ofdifferent chain lengths, for example laurate, myristate, stearate,arachidate, behenate, oleate, arachidonate, octanedioic acid,tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and thelike, polylysine, octane, or carbohydrates (dextran, cellulose, oligo-or polysaccharides. These moieties may be direct fusions with the GM-CSFvariant and may be generated by standard cloning and expressiontechniques. Alternatively, well-known chemical coupling methods may beused to attach the moieties to the GM-CSF variant of the invention.

Polynucleotides, Vectors and Host Cells

The invention also provides polynucleotides encoding the GM-CSF variantsof the invention. The polynucleotide may be a complementary deoxynucleicacid (cDNA), and may be codon optimized for expression in suitable host.Codon optimization is a well-known technology.

In some embodiments, the polynucleotide encoding the GM-CSF variantcomprises the polynucleotide sequence of SEQ ID NOs: 10, 11, 12, 14, 15,16 or 17.

The polynucleotide sequences encoding the GM-CSF variants of theinvention may be operably linked to one or more regulatory elements,such as a promoter or enhancer, that allow expression of the nucleotidesequence in the intended host cell. The polynucleotide may be cDNA.

The invention also provides a vector comprising the polynucleotideencoding the GM-CSF variant of the invention.

The invention also provides an expression vector comprising thepolynucleotide encoding the GM-CSF variant of the invention.

The invention also provides an expression vector comprising thepolynucleotide sequence of SEQ ID NO: 10.

The invention also provides an expression vector comprising thepolynucleotide sequence of SEQ ID NO: 11.

The invention also provides an expression vector comprising thepolynucleotide sequence of SEQ ID NO: 12.

The invention also provides an expression vector comprising thepolynucleotide sequence of SEQ ID NO: 13.

The invention also provides an expression vector comprising thepolynucleotide sequence of SEQ ID NO: 14.

The invention also provides an expression vector comprising thepolynucleotide sequence of SEQ ID NO: 15.

The invention also provides an expression vector comprising thepolynucleotide sequence of SEQ ID NO: 16.

The invention also provides an expression vector comprising thepolynucleotide sequence of SEQ ID NO: 17.

Such vectors may be plasmid vectors, viral vectors, vectors forbaculovirus expression, transposon based vectors or any other vectorsuitable for introduction of the synthetic polynucleotide of theinvention into a given organism or genetic background by any means. Forexample, polynucleotides encoding the GM-CSF variants of the invention,optionally conjugated to a half-life extending moiety, are inserted intoexpression vectors. The DNA segments encoding immunoglobulin chains maybe operably linked to control sequences in the expression vector(s) thatensure the expression of immunoglobulin polypeptides. Such controlsequences include signal sequences, promoters (e.g. naturally associatedor heterologous promoters), enhancer elements, and transcriptiontermination sequences, and are chosen to be compatible with the hostcell chosen to express the antibody. Once the vector has beenincorporated into the appropriate host, the host is maintained underconditions suitable for high level expression of the proteins encoded bythe incorporated polynucleotides.

Suitable expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markerssuch as ampicillin-resistance, hygromycin-resistance, tetracyclineresistance, kanamycin resistance or neomycin resistance to permitdetection of those cells transformed with the desired DNA sequences.

Suitable promoter and enhancer elements are known in the art. Forexpression in a eukaryotic cell, exemplary promoters include lightand/or heavy chain immunoglobulin gene promoter and enhancer elements,cytomegalovirus immediate early promoter, herpes simplex virus thymidinekinase promoter, early and late SV40 promoters, promoter present in longterminal repeats from a retrovirus, mouse metallothionein-I promoter,tetracycline-inducible promoter, and various art-known tissue specificpromoters. Selection of the appropriate vector and promoter is wellknown.

An exemplary promoter that can be used comprises the amino acid sequenceof SEQ ID NO: 31 and may be encoded by a polynucleotide comprising thepolynucleotide sequence of SEQ ID NO: 32.

SEQ ID NO: 31 MAWVWTLLFLMAAAQSIQA SEQ ID NO: 32ATGGCCTGGGTGTGGACCCTGCTGTTCCTGATGGCCGCCGCCCAGAGCAT CCAGGCC

Large numbers of suitable vectors and promoters are known. Many arecommercially available for generating recombinant constructs. Exemplaryvectors are bacterial vectors pBs, phagescript, PsiX174, pBluescript SK,pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif.,USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia,Uppsala, Sweden), and eukaryotic vectors pWLneo, pSV2cat, pOG44, PXR1,pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.4 (Lonza)and pEE12.4 (Lonza). Exemplary promoters include light and/or heavychain immunoglobulin gene promoter and enhancer elements,cytomegalovirus immediate early promoter, herpes simplex virus thymidinekinase promoter, early and late SV40 promoters, promoter present in longterminal repeats from a retrovirus, mouse metallothionein-I promoter,tetracycline-inducible promoter, and various art-known tissue specificpromoters. Selection of the appropriate vector and promoter is wellknown.

The invention also provides a host cell comprising one or more vectorsof the invention. “Host cell” refers to a cell into which a vector hasbeen introduced. It is understood that the term host cell is intended torefer not only to the particular subject cell but to the progeny of sucha cell, and also to a stable cell line generated from the particularsubject cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not be identical to the parent cell, but are still includedwithin the scope of the term “host cell” as used herein. Such host cellsmay be eukaryotic cells, prokaryotic cells, plant cells or archealcells. Escherichia coli, bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species are examples of prokaryotic host cells. Othermicrobes, such as yeast, are also useful for expression. Saccharomyces(e.g., S. cerevisiae) and Pichia are examples of suitable yeast hostcells. Exemplary eukaryotic cells may be of mammalian, insect, avian orother animal origins Mammalian eukaryotic cells include immortalizedcell lines such as hybridomas or myeloma cell lines such as SP2/0(American Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NSO(European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK,ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murinecell lines. An exemplary human myeloma cell line is U266 (ATTCCRL-TIB-196). Other useful cell lines include those derived from ChineseHamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologics,Walkersville, Md.), CHO-K1 (ATCC CRL-61) or DG44.

The invention also provides a method of producing the GM-CSF variant ofthe invention, comprising culturing the host cell of the invention inconditions that the GM-CSF variant is expressed, and recovering theGM-CSF variant produced by the host cell. Once synthesized (eitherchemically or recombinantly), the GM-CSF variants may be purifiedaccording to standard procedures, including ammonium sulfateprecipitation, affinity columns, column chromatography, high performanceliquid chromatography (HPLC) purification, gel electrophoresis, and thelike (see generally Scopes, Protein Purification (Springer-Verlag, N.Y.,(1982)). The GM-CSF variant of the invention may be substantially pure,e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, atleast about 90% to 95% pure, or at least about 98% to 99%, or more,pure, e.g., free from contaminants such as cell debris, macromolecules,etc. other than the GM-CSF variant of the invention.

The polynucleotides encoding the GM-CSF variants of the invention may beincorporated into vectors using standard molecular biology methods. Hostcell transformation, culture, antibody expression and purification aredone using well known methods.

Methods of Use

The GM-CSF variants of the invention have in vitro and in vivotherapeutic and prophylactic utilities. For example, the GM-CSF variantsof the invention may be administered to cells in culture, in vitro or exvivo, or to a subject to treat, prevent, and/or diagnose a variety ofdisorders, such as inflammatory bowel disease (IBD).

The invention provides a method of treating inflammatory bowel disease(IBD) in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the GM-CSF variant of theinvention for a time sufficient to treat IBD.

In some embodiments, IBD is Crohn's disease.

In some embodiments, IBD is an ulcerative colitis.

In some embodiments, IBD is Johne's disease, Behçet's syndrome,collagenous colitis, diversion colitis, indeterminate colitis,microscopic colitis, infective colitis, ischaemic colitis, lymphocyticcolitis, idiopathic inflammation of the small and/or proximal intestine,IBD-related diarrhea and closely related diseases and disorders of thegastrointestinal tract.

In some embodiments, the subject is in remission.

In some embodiments, the subject is resistant to treatment with at leastone of the therapeutics an aminosalicylate, a corticosteroid, animmunomodulator, an antibiotic, or a biologic.

The methods of the invention may be used to treat a subject belonging toany animal classification. Examples of subjects that may be treatedinclude mammals such as humans, rodents, dogs, cats and farm animals.

The GM-CSF variants of the invention may be useful in the preparation ofa medicament for such treatment, wherein the medicament is prepared foradministration in dosages defined herein.

In some embodiments, the GM-CSF variant is administered as an inductiontherapy.

In some embodiments, the GM-CSF variant is administered as a maintenancetherapy.

“Therapeutically effective amount” of the GM-CSF variant of theinvention effective in the treatment of IBD may be determined bystandard research techniques. Selection of a particular effective dosemay be determined (e.g., via clinical trials) by those skilled in theart based upon the consideration of several factors. Such factorsinclude the disease to be treated or prevented, the symptoms involved,the patient's body mass, the patient's immune status and other factorsknown by the skilled artisan. The precise dose to be employed in theformulation will also depend on the route of administration, and theseverity of disease, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses canbe extrapolated from dose-response curves derived from in vitro oranimal model test systems.

“Treat” or “treatment” refers to therapeutic treatment wherein theobject is to slow down (lessen) an undesired physiological change ordisease, or to provide a beneficial or desired clinical outcome duringtreatment. Beneficial or desired clinical outcomes include alleviationof symptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” mayalso mean prolonging survival as compared to expected survival if asubject was not receiving treatment. Those in need of treatment includethose subjects already with the undesired physiological change ordisease as well as those subjects prone to have the physiological changeor disease. An exemplary beneficial clinical outcome is to achieveremission for IBD, which may be assessed by clinical and visualexamination of the G1 tract (e.g. by endoscopy).

The GM-CSF variants of the invention may also be administered to asubject to treat, prevent, and/or diagnose autoimmune pulmonary alveolarproteinosis (aPAP). aPAP is a rare lung disease resulting from theaccumulation of surfactant protein. Surfactant homeostasis is normallymaintained by alveolar macrophages in a GM-CSF-dependent manner (Tazawa,et al., (2014). Chest, 145(4), 729-737). The cause of aPAP has beenattributed to high levels of GM-CSF autoantibodies in the lung whichlimit alveolar macrophage function. While whole lung lavage is thestandard of care for aPAP, systemic or inhaled administration of GM-CSFhas demonstrated clinical benefit to PAP patients (Seymour et al.,(2001) American Journal of Respiratory and Critical Care Medicine, 163,524-531).

Pharmaceutical Compositions and Administration

The invention also provides pharmaceutical compositions comprising theGM-CSF variants of the invention and a pharmaceutically acceptablecarrier. For therapeutic use, the GM-CSF variants of the invention maybe prepared as pharmaceutical compositions containing an effectiveamount of the antibody as an active ingredient in a pharmaceuticallyacceptable carrier. “Carrier” refers to a diluent, adjuvant, excipient,or vehicle with which the antibody of the invention is administered.Such vehicles may be liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. For example, 0.4%saline and 0.3% glycine may be used. These solutions are sterile andgenerally free of particulate matter. They may be sterilized byconventional, well-known sterilization techniques (e.g., filtration).The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, stabilizing, thickening, lubricatingand coloring agents, etc. The concentration of the GM-CSF variants ofthe invention in such pharmaceutical formulation may vary, from lessthan about 0.5%, usually to at least about 1% to as much as 15 or 20% byweight and may be selected primarily based on required dose, fluidvolumes, viscosities, etc., according to the particular mode ofadministration selected. Suitable vehicles and formulations, inclusiveof other human proteins, e.g., human serum albumin, are described, forexample, in e.g. Remington: The Science and Practice of Pharmacy,21^(st) Edition, Troy, D. B. ed., Lipincott Williams and Wilkins,Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the GM-CSF variants ofthe invention may be any suitable route that delivers the variant to thehost, such as parenteral administration, e.g., intradermal,intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary,transmucosal (oral, intranasal, intravaginal, rectal), using aformulation in a tablet, capsule, solution, powder, gel, particle; andcontained in a syringe, an implanted device, osmotic pump, cartridge,micropump; or other means appreciated by the skilled artisan, as wellknown in the art. Site specific administration may be achieved by forexample intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracerebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac,intraperitoneal, intrapleural, intraprostatic, intrapulmonary,intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial,intrathoracic, intrauterine, intravascular, intravesical, intralesional,vaginal, rectal, buccal, sublingual, intranasal, or transdermaldelivery.

The GM-CSF variants of the invention may be administered to a subject byany suitable route, for example parentally by intravenous (i.v.)infusion or bolus injection, intramuscularly or subcutaneously orintraperitoneally. i.v. infusion may be given over for example 15, 30,60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11 or 12 hours.

The dose given to a subject is sufficient to alleviate or at leastpartially arrest the disease being treated (“therapeutically effectiveamount”) and may be sometimes 0.005 mg to about 100 mg/kg, e.g. about0.05 mg to about 30 mg/kg or about 5 mg to about 25 mg/kg, or about 4mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg , or for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, forexample about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50,60, 70, 80, 90 or 100 mg/kg.

A fixed unit dose may also be given, for example, 50, 100, 200, 500 or1000 mg, or the dose may be based on the patient's surface area, e.g.,500, 400, 300, 250, 200, or 100 mg/m². Usually between 1 and 8 doses,(e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may be administered to treat thepatient, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more dosesmay be given.

The administration of the GM-CSF variants of the invention may berepeated after one day, two days, three days, four days, five days, sixdays, one week, two weeks, three weeks, one month, five weeks, sixweeks, seven weeks, two months, three months, four months, five months,six months or longer. Repeated courses of treatment are also possible,as is chronic administration. The repeated administration may be at thesame dose or at a different dose. For example, the GM-CSF variants ofthe invention may be administered at 8 mg/kg or at 16 mg/kg at weeklyinterval for 8 weeks, followed by administration at 8 mg/kg or at 16mg/kg every two weeks for an additional 16 weeks, followed byadministration at 8 mg/kg or at 16 mg/kg every four weeks by intravenousinfusion.

For example, the GM-CSF variants of the invention may be provided as adaily dosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0,1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80,90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, oralternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, orany combination thereof, using single or divided doses of every 24, 12,8, 6, 4, or 2 hours, or any combination thereof.

The GM-CSF variants of the invention may also be administeredprophylactically in order to reduce the risk of developing IBD, delaythe onset of the occurrence of an event in IBD progression, and/orreduce the risk of recurrence when IBD is in remission.

The GM-CSF variants may be lyophilized for storage and reconstituted ina suitable carrier prior to use. This technique has been shown to beeffective with conventional protein preparations and well knownlyophilization and reconstitution techniques can be employed.

Oral Administration

The GM-CSF variants of the invention may be formulated for oraladministration. The GM-CSF variants may be formulated with or withoutthose carriers customarily used in the compounding of solid dosage formssuch as tablets and capsules. For example, a capsule may be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized Additional agents can be includedto facilitate absorption of the GM-CSF variant. Diluents, flavorings,low melting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be employed.Pharmaceutical compositions for oral administration can also beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations that can be used orally also includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

The GM-CSF variant pharmaceutical composition may be also provided in anenteric coating, the enteric coating being designed to protect andrelease the pharmaceutical composition in a controlled manner within thesubject's lower gastrointestinal system, and to avoid systemic sideeffects. In addition to enteric coatings, the GM-CSF variants of theinvention may be encapsulated, coated, engaged or otherwise associatedwithin any compatible oral drug delivery system or component. Forexample, the GM-CSF variant of the invention may be provided in a lipidcarrier system comprising at least one of polymeric hydrogels,nanoparticles, microspheres, micelles, and other lipid systems.

To overcome degradation in the small intestine, the GM-CSF variant ofthe invention may be contained within a hydrogel polymer carrier. TheGM-CSF variant of the invention may be further formulated for compatibleuse with a carrier system that is designed to increase the dissolutionkinetics and enhance intestinal absorption of the GM-CSF variant. Forexample, the GM-CSF variant may be formulated into liposomes, micellesand nanoparticles to increase GI tract permeation of the GM-CSFvariants.

Various bioresponsive systems may also be combined with the GM-CSFvariant of the invention to provide a pharmaceutical agent for oraldelivery. In some embodiments, the GM-CSF variant is used in combinationwith a bioresponsive system, such as hydrogels and mucoadhesive polymerswith hydrogen bonding groups (e.g., PEG, poly(methacrylic) acid [PMAA],cellulose, Eudragit®, chitosan and alginate) to provide a therapeuticagent for oral administration.

The GM-CSF variants of the invention may be administered in combinationwith permeation enhancers that promote the transport of the GM-CSFvariants across the intestinal mucosa by increasing paracellular ortranscellular permeation. Exemplary permeation enhancers comprise along-chain fatty acid, a bile salt, an amphiphilic surfactant, and achelating agent.

Combination Therapies

The invention also provides for a method of treating IBD, comprisingadministering to a subject in need thereof the GM-CSF variant of theinvention in combination with a second therapeutic agent.

“In combination with” refers to administering of the GM-CSF variants ofthe invention described herein and a second therapeutic agentconcurrently as single agents or sequentially as single agents in anyorder. In general, each agent will be administered at a dose and/or on atime schedule determined for that agent.

The second therapeutic agent may be any known therapy for IBD, includingany agent or combination of agents that are known to be useful, or whichhave been used or are currently in use, for treatment of IBD. Suchtherapies and therapeutic agents include an aminosalicylates, acorticosteroid, an immunomodulator, an antibiotic, or a biologic.

Aminosalicylates are effective in treating mild to moderate cases of IBDas well as preventing relapses and maintaining remission. They areusually administered orally or rectally. Sulfasalazine (Azulfidine®),the first aminosalicylate to be widely used for IBD, is effective inachieving and maintaining remission in people with mild-to-moderatedisease. It delivers 5-aminosalicylic acid (5-ASA) to the intestine butcomes with disagreeable side effects in some patients, such as headache,nausea, loss of appetite, vomiting, rash, fever, and decreased whiteblood cell count. Sulfasalazine can decrease sperm production andfunction in men while they are taking the medication. It has beenassociated with pancreatitis in rare cases. The headaches, nausea, andrash are thought to be due to the release of the sulfapyridine moietythat is necessary for delivery of the 5-ASA to the intestine.

Other derivates of 5-ASA have also been synthesized. Those derivativesinclude Asacol® or Pentasa® (mesalamine), Dipentum® (olsalazine), andColazal™ (balsalazide). Local mesalamine preparations bypass the stomachto avoid early digestion, and then release close to the inflamed sectionof the bowel. Oral, delayed-release preparations such as Pentasa® andAsacol® (mesalamine) can release 5-ASA directly to the small intestineand colon, or to the ileum and/or colon, respectively. Rowasa®, an enemaformulation of mesalamine, allows the drug to be applied directly to theleft colon. Rowasa® is effective in 80% of patients withmild-to-moderate colitis that affects only the left side of the colon.Mesalamine suppositories (Canasa®) that deliver the drug directly fromthe rectum up to the sigmoid colon are effective in a high proportion ofpatients with UC limited to the rectum and the lower end of the colon.Dipentum®, an oral, delayed-release preparation of olsalazine, delivers5-ASA directly to the colon only.

As fast-acting anti-inflammatory and immunosuppressive agents,corticosteroids have been used for treating acute flare-ups of IBD forover 50 years. Since that time, these powerful agents have been themainstay of treatment for disease. Most patients notice an improvementin symptoms within days of starting corticosteroids. This group ofmedications is available in oral, rectal, and intravenous (IV) forms.Corticosteroids are not effective in preventing flare-ups and thereforeare rarely used for maintenance therapy in IBD. Since long-term useresults in side effects, these agents are recommended only forshort-term use in order to achieve remission, but they are not usedfrequently in the latter case. For people with moderate to severe activedisease, oral corticosteroids include Deltasone® (prednisone), Medrol®(methylprednisolone), and hydrocortisone. Aminosalicylates are oftentaken together with corticosteroids.

Entocort® (budesonide), an oral corticosteroid, is used to treatmild-to-moderate Crohn's disease involving the end of the smallintestine and/or the first part of the large intestine. This nonsystemicsteroid targets the intestine rather than the whole body.Corticosteroids may also be given rectally as enemas (hydrocortisone,methylprednisone, Cortenema®), foams (hydrocortisone acetate,ProctoFoam-HC®), and suppositories. Such preparations are used formild-to-moderate ulcerative colitis that is limited to the rectum orlower part of the colon. When used in combination with other therapies,these agents are also effective against more widespread disease thatstarts at the rectum. Methylprednisone and hydrocortisone are oftengiven by IV infusion to patients with severe and extensive disease.Acute IBD does not respond to corticosteroid therapy in 20-30% of casesand in 30-40% of cases with moderate to severe disease, corticosteroidscannot be abruptly discontinued without occurrence of a diseaseflare-up.

Since IBD appears to be caused by an overactive immune system,immunomodulators play an important role in the treatment of thisdisease. These drugs are used for those who have one of the followingcharacteristics: (a) side effects with corticosteroid treatment, (b)steroid-dependent disease, (c) do not respond to aminosalicylates,antibiotics, or corticosteroids, (d) perineal disease that does notrespond to antibiotics, and (e) need to maintain remission. These drugsmay be combined with a corticosteroid to speed up response during activeflares of disease.

Imuran®, Azasan® (azathioprine) and Purinethol® (6-mercaptopurine, 6-MP)are oral immunomodulators that are used to maintain remission in Crohn'sdisease and UC. Since these agents have a slow onset of action, they areusually given along with another faster-acting drugs, e.g.corticosteroids. Other immunomodulators used for IBD are Sandimmune®,Neoral® (cyclosporine A) and Prograf® (tacrolimus). Of these agents,cyclosporine A has the fastest onset of action. When given IV at highdoses, cyclosporine A is useful against active Crohn's disease. Thisdrug is effective against severe UC as is tacrolimus. The latter agentcan be used against Crohn's when corticosteroids are not effective orwhen fistulas develop. Tacrolimus may be applied topically to treatCrohn's disease of the mouth or perineal area. An option for people withCrohn's disease who do not respond to other treatments and cannottolerate other immunosuppressants is IV-administered Rheumatrex® orMexate® (methotrexate (MTX)).

Although no specific infectious agent has been identified as the causeof IBD, antibiotics are frequently used as a primary treatment.Antibiotics are effective as long-term therapy in Crohn's diseasepatients who have fistulas (between loops of intestine or betweenintestine and adjacent organs, e.g. skin) or recurrent abscesses nearthe anus. Patients whose active disease is successfully treated withantibiotics may be kept on these as maintenance therapy. Generally,antibiotics are not considered useful for those with UC; the exceptionis toxic megacolon.

The most frequently prescribed broad-spectrum antibiotics for IBD areFlagyl® (metronidazole) and Cipro® (ciprofloxacin). Metronidazole is aprimary therapy for active Crohn's and has been shown to reduce therecurrence of Crohn's for the first three months after ileum resectionsurgery. This drug is effective in managing perineal Crohn's in over 50%of cases. Ciprofloxacin, much safer than metronidazole, is commonly usedto treat active Crohn's disease. Both oral and IV metronidazole andciprofloxacin are used for IBD treatment.

Possible targets by which biologics may interfere with the body'sinflammatory response in IBD include tumor necrosis factor-alpha(TNF-α), interleukins, adhesion molecules, colony-stimulating factors,and others. Since their mechanism is targeted, biologic therapies offera distinct advantage in IBD treatment. Unlike corticosteroids, whichtend to suppress the entire immune system and thereby produce major sideeffects, biologic agents act selectively. Biologics are targeted toparticular enzymes and proteins that have already been proven defective,deficient, or excessive in people with IBD or in animal models ofcolitis. Anti-TNF agents have been used in both Crohn's disease and UC,such REMICADE® (infliximab), SIMPONI® (golimumab) and HUMIRA®(adalimumab).

Despite the above medication options for IBD, 66-75% of Crohn's patientsand 25-40% of those with UC will eventually undergo surgery. Surgery forCrohn's disease depends upon the location of the disease. If it is inthe small intestine, areas of diseased bowel may alternate with areas ofnormal bowel. The areas of active disease may narrow, formingstrictures, which can block the passage of digested food. If the lesionsare separated, strictureplasty is often used. Here, the strictured areasare widened and the small intestine is spared. Resection and anastomosismay be needed if the stricture is long or if there are multiplestrictures close to each other. Although resection may offer years ofrelief, disease can recur at or near the site of the anastomosis. Inpatients with severe Crohn's in the colon, colectomy may be done. If therectum is unaffected the end of the ileum may be rejoined to the rectum;thus, stool may be passed normally. If both the colon and rectum areinvolved, proctocolectomy with subsequent ileostomy may be performed.Fistulas and/or abscesses eventually develop in about 25% of patientswith Crohn's disease. If fistulas are unresponsive to medication, theyare removed by resection of the affected bowel followed by anastomosis.Abscesses must be drained; in some cases, this requires resection. Foryears, the standard surgery for UC has been proctocolectomy withileostomy. Now the most common procedure is restorative proctocolectomy;this allows the patient to continue to pass stool through the anus.Unlike Crohn's disease, which can recur after surgery, UC is “cured”once the colon is removed.

Kits

One embodiment of the invention is a kit comprising the GM-CSF variantof the invention.

The kit may be used for therapeutic uses.

In some embodiments, the kit comprises the GM-CSF variant of theinvention and reagents for detecting the GM-CSF variant. The kit caninclude one or more other elements including: instructions for use;other reagents, e.g., devices or other materials for preparing theGM-CSF variant for administration; pharmaceutically acceptable carriers;and devices or other materials for administration to a subject.

In some embodiments, the kit comprises the GM-CSF variant of theinvention in a container.

In some embodiments, the kit comprises the GM-CSF variant of SEQ ID NO:2.

In some embodiments, the kit comprises the GM-CSF variant of SEQ ID NO:6.

In some embodiments, the kit comprises the GM-CSF variant of SEQ ID NO:7.

In some embodiments, the kit comprises the GM-CSF variant of SEQ ID NO:8.

In some embodiments, the kit comprises the GM-CSF variant of SEQ ID NO:9.

The invention will now be described with specific, non-limitingexamples.

EXAMPLE 1 Materials and Methods Production of Human GM-CSF Variants:

DNA and expression vectors encoding various His₆-tagged variants ofGM-CSF were synthetically produced and used to transiently transfectExpi293 (HEK cells, ThermoScientific). The secreted protein was purifiedfrom cell supernatants by immobilized metal affinity chromatography andthen buffer-exchanged into 1× PBS using standard methods and used forfurther characterization. Signal sequence used was MAWVWTLLFLMAAAQSIQA(SEQ ID NO: 19).

TF-1 Proliferation Assay

5×10³ TF-1 cells (ATCC® CRL 2003™)/well were plated in 96-well plates(Costar 3603) in Assay Medium (RPMI1640—Gibco, 11875 containing 10%FBS—Gibco, 16140, 1% PenStrep—Gibco, 10378). Serial dilution of humanGM-CSF variants as well as commercially-available recombinant protein(R&D Systems: Cat #215-GM/CF as a positive control) were prepared inAssay Medium and 50 μL/well of GM-CSF titrations was added to the cells.Cells were incubated for 72 h at 37° C. in a humidified incubator with a5% CO₂ atmosphere. Cell proliferation was measured by the addition ofPromega CellTiter 96® Aqueous One Solution (20 μL/well) according tomanufacturer's protocol and incubating the cells for an additional 4 hat 37° C. The plates were shaken for 10 min at room temperature and theabsorbance at 490 nm was read on a plate reader. Raw OD_(490 nm) valueswere plotted against the concentration of recombinant human GM-CSF(rhGM-CSF) using GraphPad Prism 6.02 to determine the EC₅₀ values.

Thermal Stability Analysis by Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry was used to assess the thermalstability of the purified variants of GM-CSF. Briefly, purified proteinwas diluted to 1 mg/mL in 1× PBS and heated from 25-120° C. at a scanrate of 1° C./min using a MicroCal VP-DSC instrument. The calorimetricdata was analyzed using Origin7 (Origin Lab Corporation). The rawcalorimetric data was normalized to the sample concentration, baselinesubtracted, and finally fit to a non-2-state model of unfolding usingOrigin7 software to obtain the T_(m) value (temperature at the midpointof unfolding) and other thermodynamic parameters.

EXAMPLE 2 Design of GM-CSF Variants

GM-CSF variants were designed and subsequently characterized for theirimproved stability (conformational stability upon heating) whileretaining and/or improving their ability to induce target cellproliferation.

Mutations were designed by the analysis of the human GM-CSF crystalstructure, PDB code 2GMF (Rozwarski, Diederichs, Hecht, Boone, &Karplus, 1996). Positions for mutations were selected not to interferewith the receptor binding according to the crystal structure of theGM-CSF:receptor complex, PDB code 3CXE (Hansen et al., 2008).Additionally, the choice of L49P and R23L substitutions were supportedby a consensus GM-CSF sequence generated from the alignment of primaryamino acid sequences of the mature GM-CSF polypeptide from fiftydifferent species. Consensus design for engineering thermostability inproteins has been described previously (M. Lehmann, Pasamontes, Lassen,& Wyss, 2000; M. Lehmann, Kostrewa, et al., 2000; Martin Lehmann & Wyss,2001). FIG. 1 shows the crystal structure of the wild-type GM-CSF andthe positions of the residues considered for substitution.

Table 5 shows the generated substitutions and the rationale for eachsubstitution. Residue numbering is according to mature human GM-CSF ofSEQ ID NO: 1. GM-CSF variants with the individual substitutions andcombinations thereof were made and characterized for their cellularpotency and stability using methods described in Example 1.

SEQ ID NO: 1 APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE cDNA encoding mature WT GM-CSF SEQ ID NO: 18GCCCCCGCCCGCTCCCCCTCCCCATCGACCCAACCCTGGGAACACGTGAACGCCATTCAGGAGGCTAGGAGACTGCTGAACCTGTCCCGGGATACCGCAGCCGAGATGAACGAAACCGTGGAGGTCATCTCCGAAATGTTTGACTTGCAAGAACCTACTTGTCTGCAAACTCGCCTCGAGCTGTACAAACAGGGACTCCGGGGAAGCCTCACTAAGCTGAAGGGGCCTCTGACCATGATGGCCTCCCACTACAAGCAGCACTGCCCGCCGACGCCGGAAACCAGCTGCGCGACCCAGATCATTACCTTCGAATCGTTCAAGGAAAACCTGAAGGACTTCCTGCTTGTGATCCCGTTCGACTGCTGGGAGCCTGTGCAGGAGTAA

TABLE 5 Substitution Rationale S29C/S69C To create a novel disulfidebond that would link GM-CSF loop AB and loop BC. L49P Promote andstabilize the beta turn preceding helix B. R23L To create a leucinezipper interaction between helices A and D that would include adjacentresidues I19, L26, L110 and L114 K107I To stabilize loop AB throughhydrophobic interactions, specifically to improve interactions withadjacent residues L70 and F103.

The R23L variant has been reported by Hercus et al. (Hercus et al.,1994) to have a two-fold increase in activity in the GM-CSF-dependentproliferation of primary CML cells. However, the substitution has notbeen linked to the resulting improved conformational stability.

The K107I substitution was included to stabilize loop AB throughhydrophobic interactions with adjacent residues L70 and F103. There isprecedent in nature for this combination of hydrophobic amino acids,L70/F103/I107, which are found in rat GM-CSF.

Table 6 shows the amino acid sequences of the generated variants andTable 7 shows the cDNA sequences encoding the generated GM-CSF variants.FIG. 2 shows the amino acid sequence alignments of the generatedvariants.

TABLE 6 human GM-CSF SEQ ID Variant NO: Amino acid sequence S29C/S69C 2APARSPSPSTQPWEHVNAIQEARRLLNLCRDTAA EMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGCLTKLKGPLTMMASHYKQHCPPTPETSCATQIIT FESFKENLKDFLLVIPFDCWEPVQE L49P 3APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAA EMNETVEVISEMFDPQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIIT FESFKENLKDFLLVIPFDCWEPVQE K107I 4APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAA EMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIIT FESFIENLKDFLLVIPFDCWEPVQE R23L 5APARSPSPSTQPWEHVNAIQEALRLLNLSRDTAA EMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIIT FESFKENLKDFLLVIPFDCWEPVQES29C/S69C/L49P 6 APARSPSPSTQPWEHVNAIQEARRLLNLCRDTAAEMNETVEVISEMFDPQEPTCLQTRLELYKQGLRG CLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE S29C/S69C/K107I 7APARSPSPSTQPWEHVNAIQEARRLLNLCRDTAA EMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGCLTKLKGPLTMMASHYKQHCPPTPETSCATQIIT FESFIENLKDFLLVIPFDCWEPVQES29C/S69C/R23L/ 8 APARSPSPSTQPWEHVNAIQEALRLLNLCRDTAA L49P/K107IEMNETVEVISEMFDPQEPTCLQTRLELYKQGLRG CLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFIENLKDFLLVIPFDCWEPVQE S29C/S69C/L49P/ 9APARSPSPSTQPWEHVNAIQEARRLLNLCRDTAA K107IEMNETVEVISEMFDPQEPTCLQTRLELYKQGLRG CLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFIENLKDFLLVIPFDCWEPVQE

TABLE 7 human GM-CSF SEQ ID Variant NO: cDNA sequence S29C/S69C 10GCCCCCGCCCGCTCCCCCTCCCCATCGACCCAA CCCTGGGAACACGTGAACGCCATTCAGGAGGCTAGGAGACTGCTGAACCTGTGCCGGGATACCG CAGCCGAGATGAACGAAACCGTGGAGGTCATCTCCGAAATGTTTGACTTGCAAGAACCTACTTGT CTGCAAACTCGCCTCGAGCTGTACAAACAGGGACTCCGGGGATGTCTCACTAAGCTGAAGGGGC CTCTGACCATGATGGCCTCCCACTACAAGCAGCACTGCCCGCCGACGCCGGAAACCAGCTGCGC GACCCAGATCATTACCTTCGAATCGTTCAAGGAAAACCTGAAGGACTTCCTGCTTGTGATCCCGT TCGACTGCTGGGAGCCTGTGCAGGAGTGATAA L49P11 GCCCCCGCCCGCTCCCCCTCCCCATCGACCCAA CCCTGGGAACACGTGAACGCCATTCAGGAGGCTAGGAGACTGCTGAACCTGTCCCGGGATACCG CAGCCGAGATGAACGAAACCGTGGAGGTCATCTCCGAAATGTTTGACCCACAAGAACCTACTTGT CTGCAAACTCGCCTCGAGCTGTACAAACAGGGACTCCGGGGAAGCCTCACTAAGCTGAAGGGGC CTCTGACCATGATGGCCTCCCACTACAAGCAGCACTGCCCGCCGACGCCGGAAACCAGCTGCGC GACCCAGATCATTACCTTCGAATCGTTCAAGGAAAACCTGAAGGACTTCCTGCTTGTGATCCCGT TCGACTGCTGGGAGCCTGTGCAGGAGTGATAA K107I12 GCCCCCGCCCGCTCCCCCTCCCCATCGACCCAA CCCTGGGAACACGTGAACGCCATTCAGGAGGCTAGGAGACTGCTGAACCTGTCCCGGGATACCG CAGCCGAGATGAACGAAACCGTGGAGGTCATCTCCGAAATGTTTGACTTGCAAGAACCTACTTGT CTGCAAACTCGCCTCGAGCTGTACAAACAGGGACTCCGGGGAAGCCTCACTAAGCTGAAGGGGC CTCTGACCATGATGGCCTCCCACTACAAGCAGCACTGCCCGCCGACGCCGGAAACCAGCTGCGC GACCCAGATCATTACCTTCGAATCGTTCATCGAAAACCTGAAGGACTTCCTGCTTGTGATCCCGTT CGACTGCTGGGAGCCTGTGCAGGAGTGATAA R23L13 GCCCCCGCCCGCTCCCCCTCCCCATCGACCCAA CCCTGGGAACACGTGAACGCCATTCAGGAGGCTCTTAGACTGCTGAACCTGTCCCGGGATACCGC AGCCGAGATGAACGAAACCGTGGAGGTCATCTCCGAAATGTTTGACTTGCAAGAACCTACTTGTC TGCAAACTCGCCTCGAGCTGTACAAACAGGGACTCCGGGGAAGCCTCACTAAGCTGAAGGGGCC TCTGACCATGATGGCCTCCCACTACAAGCAGCACTGCCCGCCGACGCCGGAAACCAGCTGCGCG ACCCAGATCATTACCTTCGAATCGTTCAAGGAAAACCTGAAGGACTTCCTGCTTGTGATCCCGTT CGACTGCTGGGAGCCTGTGCAGGAGTGATAAS29C/S69C/L49P 14 GCCCCCGCCCGCTCCCCCTCCCCATCGACCCAACCCTGGGAACACGTGAACGCCATTCAGGAGGC TAGGAGACTGCTGAACCTGTGCCGGGATACCGCAGCCGAGATGAACGAAACCGTGGAGGTCATC TCCGAAATGTTTGACCCACAAGAACCTACTTGTCTGCAAACTCGCCTCGAGCTGTACAAACAGGG ACTCCGGGGATGTCTCACTAAGCTGAAGGGGCCTCTGACCATGATGGCCTCCCACTACAAGCAG CACTGCCCGCCGACGCCGGAAACCAGCTGCGCGACCCAGATCATTACCTTCGAATCGTTCAAGG AAAACCTGAAGGACTTCCTGCTTGTGATCCCGTTCGACTGCTGGGAGCCTGTGCAGGAGTGATAA S29C/S69C/ 15GCCCCCGCCCGCTCCCCCTCCCCATCGACCCAA K1071 CCCTGGGAACACGTGAACGCCATTCAGGAGGCTAGGAGACTGCTGAACCTGTGCCGGGATACCG CAGCCGAGATGAACGAAACCGTGGAGGTCATCTCCGAAATGTTTGACTTGCAAGAACCTACTTGT CTGCAAACTCGCCTCGAGCTGTACAAACAGGGACTCCGGGGATGTCTCACTAAGCTGAAGGGGC CTCTGACCATGATGGCCTCCCACTACAAGCAGCACTGCCCGCCGACGCCGGAAACCAGCTGCGC GACCCAGATCATTACCTTCGAATCGTTCATCGAAAACCTGAAGGACTTCCTGCTTGTGATCCCGTT CGACTGCTGGGAGCCTGTGCAGGAGTGATAAS29C/S69C/ 16 GCCCCCGCCCGCTCCCCCTCCCCATCGACCCAA R23L/L49P/CCCTGGGAACACGTGAACGCCATTCAGGAGGC K107I TTTGAGACTGCTGAACCTGTGCCGGGATACCGCAGCCGAGATGAACGAAACCGTGGAGGTCATC TCCGAAATGTTTGACCCACAAGAACCTACTTGTCTGCAAACTCGCCTCGAGCTGTACAAACAGGG ACTCCGGGGATGCCTCACTAAGCTGAAGGGGCCTCTGACCATGATGGCCTCCCACTACAAGCAG CACTGCCCGCCGACGCCGGAAACCAGCTGCGCGACCCAGATCATTACCTTCGAATCGTTCATCGA AAACCTGAAGGACTTCCTGCTTGTGATCCCGTTCGACTGCTGGGAGCCTGTGCAGGAGTGATAA S29C/S69C/ 17GCCCCCGCCCGCTCCCCCTCCCCATCGACCCAA L49P/K107ICCCTGGGAACACGTGAACGCCATTCAGGAGGC TAGGAGACTGCTGAACCTGTGCCGGGATACCGCAGCCGAGATGAACGAAACCGTGGAGGTCATC TCCGAAATGTTTGACCCACAAGAACCTACTTGTCTGCAAACTCGCCTCGAGCTGTACAAACAGGG ACTCCGGGGATGTCTCACTAAGCTGAAGGGGCCTCTGACCATGATGGCCTCCCACTACAAGCAG CACTGCCCGCCGACGCCGGAAACCAGCTGCGCGACCCAGATCATTACCTTCGAATCGTTCATCGA AAACCTGAAGGACTTCCTGCTTGTGATCCCGTTCGACTGCTGGGAGCCTGTGCAGGAGTGATAA

Table 8 shows the summary of thermal stability of human GM-CSF variants,measured using methods described in Example 1 and expressed as themelting temperature T. and shift in the T. when compared to thewild-type GM-CSF protein. All variants exhibited significantly improvedthermal stability when compared to the wild-type protein. From theindividual substitutions, introduction of the disulfide bridge in theS29C/S69C into the wild-type GM-CSF resulted in the most enhancedstabilization when compared to the L49P, K107Iand R23L substitutionsalone. Introduction of variants combinatorially further improved thethermal stability of the resulting variant in an approximately additivemanner The variant containing all five amino acid substitutionsdescribed above, S29C/S69C/R23L/L49P/K107I, had a T_(m) value that wasmore than 28° C. greater than that of the wild-type protein.

TABLE 8 Thermal stability Protein T_(m) (° C.) ΔT_(m) (° C.)* Wild type67.06 0.00 S29C/S69C 80.86 13.80 L49P 73.84 6.78 K107I 73.95 6.89 R23L71.64 4.58 S29C/S69C/L49P 87.47 20.41 S29C/S69C/K107I 86.09 19.03S29C/S69C/R23L/L49P/K107I 95.27 28.21 S29C/S69C/L49P/K107I 92.49 25.43*relative to the wild type protein

The resulting variants were tested for their potency in a TF-1 cellproliferation assay. Table 9 shows the EC₅₀ values for each variantexpressed as mean of two individual measurements and the fold changewhen compared to the wild-type GM-CSF. The S29C/S69C demonstrated about2.6-fold improvement when compared to the wild-type GM-CSF. Theindividual substitutions L49P and R23L had a modest improvement inpotency whereas the K107I substitution resulted in a variant withslightly reduced potency. Generally, increases in the cellular potencyof the GM-CSF variants correlated with the conformation stability of thevariants with the most stable variant (S29C/S69C/R23L/L49P/K107I)exhibiting the greatest enhancement (4-fold) in stimulating theproliferation of TF-1 cells.

TABLE 9 EC₅₀ Fold-EC₅₀ Protein (pM) change* Wild-type GM-CSF 13.095 1.00S29C/S69C 5.0235 2.61 L49P 12.54 1.04 K107I 24.535 0.53 R23L 11.848 1.11S29C/S69C/L49P 10.141 1.29 S29C/S69C/K107I 3.8145 3.43S29C/S69C/R23L/L49P/K107I 3.618 3.62 S29C/S69C/L49P/K107I 8.514 1.54*Relative to the wild-type GM-CSF

EXAMPLE 3 GM-CSF Variants are Stabile in Fasted State SimulatedIntestinal Fluid

Orally-administered GM-CSF for local delivery could prove more desirablefrom a patient compliance as well as from a safety perspective byminimizing systemic exposure. However, the oral delivery of protein tothe lower gastrointestinal tract presents numerous challenges owing tothe harsh pH, proteolytic, and microbial environments to which thebiologic drug would be exposed (Amidon, Brown, & Dave, 2015).

Stability of the generated GM-CSF variants were tested in fasted statesimulated intestinal fluids (FaSSIF) supplemented with pancreatin (3mg/mL; trypsin, amylase, lipase, ribonuclease, and other proteases) inorder to assess their stability in an environment comparable to portionsof the GI tract.

FaSSIF-V2 (Biorelevant; London, UK) was prepared fresh according to themanufacturer's specifications and supplemented with pancreatin fromporcine pancreas (Sigma; St. Louis, Mo., USA) at a final concentrationof 3 mg/mL. GM-CSF variants formulated in 1× PBS were diluted to a finalconcentration of 1 mg/mL with FaSSIF (containing pancreatin) andincubated at 37° C. for 0-30 min. The digestion was arrested by heatingthe samples at 95° C. for 5 min followed by freezing. SDS-PAGE analysisof the samples was performed by loading 10 μg of GM-CSF (based onpre-treatment concentration) per lane. The resulting gel was analyzed bydensitometry to quantitate the amount of intact variant GM-CSFremaining, which was expressed as a percentage of the untreated control.

All tested variants except the R23L variant (data not shown)demonstrated improved stability to proteolytic degradation over timewhen compared to the wild type GM-CSF. FIG. 3 shows the stability ofS29C/S69C, L49L, S29C/S69C/K107I and S29C/S69C/R23L/L49P/K107I GM-CSFvariants over time in FaSSIF containing 3 mg/mL pancreatin. Compared tothe wild type GM-CSF which is completely degraded in less than oneminute in FaSSIF containing pancreatin, more than half of theS29C/S69C/R23L/L49P/K107I variant protein remains intact after 30minutes of enzymatic exposure.

EXAMPLE 4 In Silico Assessment of Immunogenicity Risk of Select GM-CSFVariants

Two GM-CSF variants were subjected to in silico analysis to determine ifany of the amino acid substitutions (relative to wild-type GM-CSF) wouldbe predicted to increase the binding affinity of any 9-mer peptides toclass II MHC molecules and therefore predict T cell epitopes usingImmunoFilter™ Technology. No major potential immunogeneicity liabilitieswere identified in the variants tested (S29C/S69C/R23L/L49P/K107I andS29C/S69C/L49P/K107I variants).

The L49P amino acid substitution appeared to reduce the immunogenicityrisk. The predicted binding scores of one 9-mer with the L49Psubstitution to HLA-DR1, HLA-DR3, HLA-DR4 and HLA-DR5 was reduced from24-32% to 4-8%.

EXAMPLE 5 GM-CSF Variants Retain their Functional Activity in FastedState Simulated Intestinal Fluid (FaSSIF)

Biological activity of GM-CSF variants R23L/S29C/L49P/S69C/K107I andS29C/L49P/S69C/K107I was assessed after exposure to FaSSIF supplementedwith trypsin, amylase and lipase, ribonuclease, and other proteases,produced by exocrine cells of the porcine pancreas proteases andribonucleases at various time periods as indicated below and in FIG. 4A,FIG. 4B, FIG. 4C and FIG. 4D.

Both variants R23L/S29C/L49P/S69C/K107I and S29C/L49P/S69C/K107Iretained their activity completely or partially at 30 minutes (FIG. 4A),1 hour (FIG. 4B) and 4 hours (FIG. 4C) of exposure to the proteolyticenvironment mimicking the GI tract, whereas the wild-type GM-CSF wascompletely inactive after 30 minutes of exposure (FIG. 4A). The variantR23L/S29C/L49P/S69C/K107I retained a substantial portion of itsbiological activity even after 6 hours of exposure to the proteolyticenvironment (FIG. 4D). Both variants were more potent in inducing STAT5phosphorylation when compared to the wild-type GM-CSF even innon-proteolytic environment (incubation in the absence of FaSSIF).

Methods

Variants of human GM-CSF were diluted to a final concentration of 1mg/mL in simulated intestinal fluid (prepared from FaSSIF-v2 powder;Biorelevant; London, UK), which was supplemented with porcine pancreatin(Sigma; St. Louis, Mo.) at a final concentration of 3 mg/mL. Variants ofhuman GM-CSF were incubated in this solution at 37° C. for 0.5-6 hours.Proteolytic digestion was arrested by the addition of complete proteaseinhibitor (Roche) to 10×. Simulated intestinal fluid-treated GM-CSFsamples were serially diluted twofold in RPMI1640 serum-free media(Gibco) and applied to TF-1 cells (ATCC; 1e5 cells/well), which had beenserum starved for 2 hours at 37° C. with 5% CO₂. Treated TF-1 cells wereincubated for 15 min at 37° C. The TF-1 cells were collected bycentrifugation and lysed with Tris lysis buffer containing protease andphosphatase inhibitors (Meso Scale Discovery). Phosphorylation of STAT5(Tyr694) relative to total STAT5a,b was determined by immunoassay (MesoScale Discovery). Phosphorylation of STAT5 protein (% relative to totalSTAT5a,b) was plotted as a function of GM-CSF concentration.

EXAMPLE 6 GM-CSF Variants Retain their Functional Activity Upon Exposureto Colon Content

Biological activity of GM-CSF variants R23L/S29C/L49P/S69C/K107I andS29C/L49P/S69C/K107I was assessed after exposure to colon content fromnaïve cynomolgus monkeys at various time periods.

FIG. 5A shows that biological activity of the GM-CSF variantsR23L/S29C/L49P/S69C/K107I and S29C/L49P/S69C/K107I was fully retainedafter incubation for 30 minutes with colon content from naive cynomolgusmonkeys, whereas the biological activity of the wild-type GM-CSF wasalmost completely abolished.

FIG. 5B shows that biological activity of the GM-CSF variantsR23L/S29C/L49P/S69C/K107I and S29C/L49P/S69C/K107I was retained afterincubation for 2 hours with colon content from naive cynomolgus monkeys.The R23L/S29C/L49P/S69C/K107I variant of GM-CSF exhibited only a twofoldloss in activity after exposure to colon content for two hours and stillpossessed potency that was twofold greater than wild-type GM-CSF thatwas not exposed to colon content. The S29C/L49P/S69C/K107I variant ofGM-CSF exhibited a 6-fold loss of activity compared to the untreatedcytokine.

FIG. 5C that shows that biological activity of the GM-CSF variantR23L/S29C/L49P/S69C/K107I was retained after incubation for 6 hours withcolon content from naive cynomolgus monkeys. TheR23L/S29C/L49P/S69C/K107I variant of GM-CSF exhibited a 5-fold loss ofactivity compared to the untreated variant cytokine while the activityof wild-type GM-CSF was completely abolished.

FIG. 5D shows that biological activity of GM-CSF and its variantsS29C/L49P/S69C/K107I and R23L/S29C/L49P/S69C/K107I was abolished afterincubation for 24 hours with colon content from naive cynomolgusmonkeys.

Table 10 shows the EC₅₀ values in the functional assay for the variants.

TABLE 10 EC₅₀ (pM) Incubation S29C/L49P/ R23L/S29C/L49P/ time WTS69C/K107I S69C/K107I Colon GM-CSF variant variant content − + − + − +0.5 hrs   50 2,700 18 25 19 17 2 hrs 120 ND 60 360 22 39 6 hrs 69 ND 51ND 35 190 24 hrs  410 ND 135 ND 70 ND ND = No activity detected; WT:wild-type

Materials and Methods

Variants of human GM-CSF were diluted to a final concentration of 1mg/mL in either 1× phosphate-buffered saline or colon content from naivecynomolgus monkeys (BioreclamationlVT; Long Island, N.Y.), which wasnormalized to a final protein concentration of 3 mg/mL in 1×phosphate-buffered saline. Variants of human GM-CSF were incubated inthis solution at 37° C. for 0.5-24 hours. After the indicated incubationperiod, GM-CSF containing samples were serially diluted twofold inRPMI1640 serum-free media (Gibco) and applied to TF-1 cells (ATCC; 1e5cells/well), which had been serum starved for 2 hours at 37° C. with 5%CO₂. Treated TF-1 cells were incubated for 15 min at 37° C. The TF-1cells were collected by centrifugation and lysed with Tri lysis buffer(Meso Scale Discovery; Rockville, Md.) containing protease andphosphatase inhibitors (Roche Life Science). Phosphorylation of STAT5(Tyr694) relative to total STAT5a,b was determined by immunoassay (MesoScale Discovery). Phosphorylation of STAT5 (% relative to totalSTAT5a,b) was plotted as a function of GM-CSF concentration. In theexperiments, wild-type GM-CSF and S29C/L49P/S69C/K107I variant had a6×-His tag at the C-terminus coupled to GM-CSF via a GS linker.

EXAMPLE 7 GM-CSF Variants Retain their Functional Activity in DifferentCanine Simulated Small Intestinal Fluid (SSIF)

The ability of wild-type human GM-CSF and GM-CSF variantR23L/S29C/L49P/S69C/K107I to stimulate STAT5 phosphorylation in TF-1cells was assessed after a two-hour exposure to SSIF supplemented withtrypsin, amylase and lipase, ribonuclease, and other proteases, producedby exocrine cells of the porcine pancreas proteases and ribonucleases asindicated in Table 11.

The functional activity of His-tagged wild-type human GM-CSF wascompletely abolished following a two-hour exposure to any of the fourpreparations of canine SSIF. Variant R23L/S29C/L49P/S69C/K107I retainedcomplete stimulatory activity after a two-hour exposure when SSIF wasprepared at pH 7 and supplemented with 1 mg/mL pancreatin, which isequivalent to 200 USP units of protease activity per milliliter Whenexposed to ten times more pancreatin (10 mg/mL; 2,000 USP units/mL) atpH 7.0 over the same timeframe, R23L/S29C/L49P/S69C/K107I GM-CSFexhibited a 24-fold loss in potency relative to variant GM-CSF notexposed to SSIF. Variant R23L/S29C/L49P/S69C/K107I exhibited a 5-foldloss in functional activity after a two-hour exposure to canine SSIFwhen the simulated fluid was prepared at pH 5.5 and supplemented with 1mg/mL pancreatin, which is equivalent to 200 USP units of proteaseactivity per milliliter When exposed to ten times more pancreatin (10mg/mL; 2,000 USP units/mL) at pH 5.5, R23L/S29C/L49P/S69C/K107I GM-CSFexhibited a 47-fold loss in potency relative to the non-pretreatedsample. Even in the absence of pre-treatment with canine SSIF,R23L/S29C/L49P/S69C/K107I GM-CSF was two and a half times more potentthan wild-type GM-CSF at inducing STAT5 phosphorylation in TF-1 cells.

TABLE 11 EC₅₀ value (pM) [Pancreatin] S29C/S69C/R23L/L49P/K107I (mg/mL)pH WT GM-CSF variant 10 5.5 No Activity 166 10 7.0 No Activity 85.2 15.5 No Activity 16.4 1 7.0 No Activity 2.94 0 (control) 7.2 8.68 3.55

Methods

Variants of human GM-CSF were diluted to a final concentration of 1mg/mL in simulated intestinal fluid (prepared from Dog FaSSIF/Dog FaSSGFpowder at a final pH of either 5.5 or 7; Biorelevant; London, UK), whichwas supplemented with porcine pancreatin (Sigma Catalog P7545; St.Louis, Mo.) at a final concentration of either 1 or 10 mg/mL. Variantsof human GM-CSF were incubated in this solution at 37° C. for 2 hours.Proteolytic digestion was arrested by the addition of complete proteaseinhibitor (Roche) to 10×. Simulated intestinal fluid-treated GM-CSFsamples were serially diluted twofold in RPMI1640 serum-free media(Gibco) and applied to TF-1 cells (ATCC; 1e5 cells/well), which had beenserum starved for 2 hours at 37° C. with 5% CO₂. Treated TF-1 cells wereincubated for 15 min at 37° C. The TF-1 cells were collected bycentrifugation and lysed with Tris lysis buffer containing protease andphosphatase inhibitors (Meso Scale Discovery). Phosphorylation of STAT5(Tyr694) relative to total STAT5a,b was determined by immunoassay (MesoScale Discovery). Phosphorylation of STAT5 protein (% relative to totalSTAT5a,b) was plotted as a function of GM-CSF concentration.Curve-fitting was performed in Prism 7 (GraphPad) to obtain the EC₅₀values.

We claim:
 1. An isolated GM-CSF variant comprising an amino acidsequence of SEQ ID NO:
 33. 2. The GM-CSF variant of claim 1, wherein thevariant exhibits at least about 5° C. higher melting temperature (T_(m))when compared to that of the wild-type GM-CSF having the amino acidsequence of SEQ ID NO:1, wherein the T_(m) is measured usingdifferential scanning calorimetry using a protocol described inExample
 1. 3. The GM-CSF variant of claim 1, wherein the variantstimulates proliferation of TF-1 ATCC® CRL 2003™ cells with an EC₅₀value that is at least about 1.5-fold less when compared to the EC₅₀value of stimulation of proliferation of the TF-1 ATCC® CRL 2003™ cellswith the wild-type GM-CSF using a protocol described in Example
 1. 4.The GM-CSF variant of claim 1, comprising an amino acid sequence of SEQID NO:
 8. 5. The GM-CSF variant of claim 1, comprising an amino acidsequence of SEQ ID NO:
 9. 6. The GM-CSF variant of claim 1, comprisingan amino acid sequence of SEQ ID NO:
 2. 7. The GM-CSF variant of claim1, comprising an amino acid sequence of SEQ ID NO:
 6. 8. The GM-CSFvariant of claim 1, comprising an amino acid sequence of SEQ ID NO: 7.9. An isolated GM-CSF variant comprising an amino acid sequence of SEQID NO:
 8. 10. An isolated GM-CSF variant comprising an amino acidsequence of SEQ ID NO:
 9. 11. The GM-CSF variant of claim 1, wherein thevariant is encoded by a synthetic polynucleotide of SEQ ID NOs: 10, 14,15, 16 or
 17. 12. The GM-CSF variant of claim 1, wherein the variant isconjugated to a half-life extending moiety.
 13. The GM-CSF variant ofclaim 12, wherein the half-life extending moiety is a human serumalbumin (HSA) or variants thereof, an antibody Fc region or fragmentthereof, an albumin-binding domain or a polyethylene glycol.
 14. TheGM-CSF variant of claim 12, wherein the half-life extending moiety isconjugated to the GM-CSF variant via a linker.
 15. The GM-CSF variant ofclaim 14, wherein the linker comprises the amino acid sequence of SEQ IDNOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or
 30. 16. An isolatedGM-CSF variant comprising an amino acid sequence of SEQ ID NOs: 2, 3, 4,6, 7, 8 or
 9. 17. An isolated polynucleotide a. encoding the GM-CSFvariant of claim 16; and/or b. comprising the polynucleotide sequence ofSEQ ID NOs: 10, 11, 12, 14, 15, 16 or
 17. 18. A vector comprising thepolynucleotide of claim
 17. 19. The vector of claim 18, wherein thevector is an expression vector.
 20. A host cell comprising the vector ofclaim
 19. 21. A method of producing a GM-CSF variant, comprisingculturing the host cell of claim 20 under conditions that the GM-CSFvariant is expressed, and purifying the expressed GM-CSF variant.
 22. Akit comprising the GM-CSF variant of claim
 16. 23. A pharmaceuticalcomposition comprising the GM-CSF variant of claim 16 and apharmaceutically acceptable excipient.
 24. A method of treating aninflammatory bowel disease (IBD) in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of the GM-CSF variant of claim 1 for a time sufficient to treatthe IBD.
 25. The method of claim 24, wherein the IBD is Johne's disease,Behçet's syndrome, collagenous colitis, diversion colitis, indeterminatecolitis, microscopic colitis, infective colitis, ischaemic colitis,lymphocytic colitis, idiopathic inflammation of the small and/orproximal intestine or IBD-related diarrhea.
 26. The method of claim 24,wherein the IBD is Crohn's disease or ulcerative colitis.
 27. The methodof claim 24, further comprising administering to the subject a secondtherapeutic agent.
 28. The method of claim 27, wherein the secondtherapeutic agent is an aminosalicylate, a corticosteroid, animmunomodulator, an antibiotic, or a biologic.
 29. The method of claim24, wherein the subject is in remission.
 30. The method of claim 24,wherein the GM-CSF variant is administered orally.